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This is the peer reviewed version of the following article:
Macken, A. , Lillicrap, A. and Langford, K. (2015), Benzoylurea pesticides used as veterinary medicines in aquaculture: Risks and developmental effects on nontarget crustaceans. Environ Toxicol Chem, 34: 1533-1542, which has been
published in final form at https://doi.org/10.1002/etc.2920. This article may be used for non-commercial purposes in accordance with Wiley Terms and
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Running Head: Environmental risks of veterinary medicines in aquaculture 1
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Corresponding author:
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Name: Dr. Ailbhe Macken, 4
Address: Ecotoxicology and Risk Assessment 5
Norwegian Institute for Water Research 6
Gaustadalléen, NO-0349 Norway 7
Phone: +47 98215472 8
Email: ama@niva.no 9
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Benzoylurea pesticides used as veterinary medicines in aquaculture: Risks and developmental 12
effects on non-target crustaceans.
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Ailbhe Macken†, Adam Lillicrap†, Katherine Langford‡
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†Ecotoxicology and Risk Assessment, Norwegian Institute of Water Research, Oslo, Norway.
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‡ Analytical chemistry, Norwegian Institute of Water Research, Oslo, Norway.
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3 Conflict of Interest Reporting
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The authors have no conflict of interest to declare.
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Corresponding author email: ama@niva.no 38
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Abstract: Diflubenzuron and teflubenzuron are benzoylureas that are used in aquaculture to control 39
sea lice. Flubenzurons have low toxicity to many marine species such as fish and algae, but by their 40
nature are likely to have significant adverse effects on non-target species such as crustaceans and 41
amphipods. Although the exact mechanism of toxicity is not known, these compounds are thought to 42
inhibit the production of the enzyme chitin synthase during moulting of immature stages of 43
arthropods. These chitin synthesis inhibitors are effective against the larval and pre-adult life stages 44
of sea lice. Due to their low solubility and results of recent monitoring studies conducted in Norway, 45
the sediment compartment is considered the most likely reservoir for these compounds and possible 46
remobilization from the sediment to benthic crustaceans could be of importance. For this reason, the 47
epibenthic copepod, Tisbe battagliai, was selected for use in the investigations into acute and 48
developmental effects of these compounds. For comparative purposes, azamethiphos was 49
investigated to identify differences in sensitivity and act as a negative control for developmental 50
effects at environmentally relevant concentrations. Standard acute studies with adult copepods 51
showed little or no acute toxicity at mg/L levels with the flubenzurons, while a naupliar 52
developmental test demonstrated that environmentally relevant concentrations (e.g. ng/L) caused a 53
complete cessation of moulting and finally death in the exposed copepods.
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Keywords: Tisbe battagliai, Diflubenzuron, Teflubenzuron, Azamethiphos, Naupliar development 55
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5 INTRODUCTION 57
Aquaculture in Norway has been in existence as far back as 1850, when brown trout were 58
first reared. The modern aquaculture industry in Norway started around 1970 after the construction 59
of the first seawater cages [1], which proved more successful than the land based/onshore tanks that 60
were used previously. Now the aquaculture industry in Norway is one of the most important 61
industries in coastal areas and Norway’s largest export after oil and gas, specifically commercial 62
Atlantic salmon (Salmo salar L.). The latest figures from the Norwegian Seafood Council show record 63
values for Norwegian salmon exports, with the value for the first half of 2013 totalling NOK 17.4 64
billion (ca. €2.1 billion)[2].
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As the aquaculture industry is so important economically, possible threats, such as parasitic 66
infections from sea lice (Copepoda, Caligidae), especially Lepeophtheirus salmonis and Celigus spp.
67
are important to control. Medicinal products have been employed in the aquaculture industry to 68
tackle the issue of sea lice infestation in farmed salmon since the 1980s [3]. In Norway, several 69
products are regularly employed against sea lice. For example diflubenzuron (DIF) and teflubenzuron 70
(TEF) which are benzoylurea medicines administered in fish food. On the Norwegian market they are 71
sold as Releeze vet. (0.6 g/kg DIF) and Ektoban vet. (2 g/kg TEF). In the late 1990s these two products 72
were in use, but by the end of the 1990s a voluntary ban was introduced due to the suspected 73
adverse environmental impacts of these products [4]. However, in recent years the use of these 74
products has again increased due to the resistance of the sea lice to the flubenzuron replacement 75
product emamectin benzoate [5]. From 2010 to 2011 the use of flubenzurons sharply decreased, 76
however in 2012 their usage was back up to the 2010 levels. Recently, it has also been reported that 77
the use of TEF and DIF have doubled both from 2011 to 2012 and again from 2012 to 2013 [6]. In the 78
past two years, there have been two different reports [4, 7] showing the negative impacts of 79
flubenzurons on various crustaceans. Therefore the recent increase in their use in the aquaculture 80
industry in Norway is potentially a very serious environmental situation.
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Another important veterinary medicine used in salmon farming in Norway is azamethiphos 82
(AZA) (market name Salmosan) which is a broad spectrum organophosphorus insecticide used 83
against flies and other arthropods in agriculture as well as sea lice in salmon farming. Unlike the 84
flubenzuron products, AZA is applied topically through bath treatments (with an application rate of 85
0.1 - 0.2 mg/L). The use of AZA as a potential delousing agent for salmon lice was first reported by 86
Roth and Richards [8]. The safety of azamethiphos has previously been evaluated for the 87
establishment of Maximum Residue Limits (MRLs) in Salmonidae and the conclusion was that no 88
MRLs were required for the protection of consumer safety as the measured residues in salmon 89
muscle and skin were always below the limit of detection (LOD) even one hour after treatment [9].
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The inclusion of AZA in this study was based on the reported usage of the various delousing 91
treatments used in Norway over the past few years. As the possible risks to non-target species and 92
possible persistence of the flubenzurons became of more interest, the use of AZA was seen to 93
increase in the period of 2008-2011, when the use of the flubenzurons decreased (Table 1). All three 94
compounds are widely used in salmon aquaculture in Norway at present and are of fundamental 95
importance to the Norwegian aquaculture market but also specifically to the environment.
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The Ministry of Fisheries is the main authority responsible for the licensing of new farms and 97
the control of the industry. In Norway there is mandatory sea lice monitoring, reporting and auditing.
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A condition for receiving a licence for aquaculture activities is that the activity does not comprise a 99
danger for the spreading of fish disease. The management plans for the facility must be approved by 100
the Animal Health Authority. Records must be kept of disease outbreaks, diagnoses, testing and 101
treatment and the facility must post public notice of antibiotic use.
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The licensing requirements for new medicines in Norway are very strict and comply fully with 103
EC regulations. The pharmaceuticals for treating salmon lice are approved by the Norwegian 104
Medicines Agency (NoMA) in accordance with the EU rules for the approval of veterinary medicine 105
products. The Norwegian Medicines Agency is the national regulatory authority for new and existing 106
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medicines and is responsible for all areas of medicinal products from production, trials and marketing 107
to monitoring their use. Only approved veterinarians and fish health biologists are allowed to 108
prescribe medicines for use on fish. In addition there are also regulations specifying the length of 109
time necessary between treatments with veterinary products and slaughtering of the fish. The 110
Norwegian Food Safety Authority is responsible for keeping a record of all pharmaceuticals ordered 111
for use in the aquaculture industry in Norway.
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The benzoylurea pesticides are not very toxic to many marine species such as fish and algae, 113
but due to their specific mode of action (MOA) they are likely to have adverse effects on non-target 114
species such as the crustaceans and amphipods in the marine environment [4]. Although the exact 115
mechanism of toxicity is not known, these compounds are thought to inhibit the expression of the 116
enzyme chitin synthase during moulting in immature stages of arthropod development. These chitin 117
synthesis inhibitors are therefore effective against the larval and pre-adult life stages of sea lice. The 118
life cycle of sea lice have been extensively researched [10] and is characterized by 3 distinct 119
morphological life stages (naupliar, copepodid and chalimus). The number of moults per life stage 120
can vary between species of sea lice but the free living stage is generally characterized by 2 naupliar 121
stages and the infective copepod stages [10]. Following attachment to the host fish, the sea lice 122
moult into a number of chalimus stages before becoming adult.
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Due to the relatively low solubility of the flubenzuron compounds (9.4 and 89 µg/L 124
for TEF and DIF respectively), they are administered to the fish via the food and consequently 125
uneaten food and faeces are expected to be the main routes into the environment. In combination 126
with previous monitoring studies conducted recently in Norway [4], it was concluded that the 127
sediment is the most likely reservoir for these compounds and that possible remobilization from the 128
sediment to benthic living crustaceans could be of ecological importance. For this reason, the 129
epibenthic copepod Tisbe battagliai was selected to investigate the developmental effects of these 130
compounds to a non-target species. T. battagliai is a sexually reproducing marine harpacticoid 131
copepod commonly used in the ecotoxicological assessment of chemicals and pollutants.
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Harpacticoids are members of the benthic fauna with a few planktonic species living in close 133
association with other organisms. They often represent the second largest meiofaunal group in 134
marine sediments after nematodes [11]. Therefore, they are highly representative of non-target 135
species that may be exposed in and around fish farms treated with veterinary medicines.
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Development through the six naupliar stages to the copepodite stage C1 typically occurs within 96 h 137
under optimal conditions (e.g. food quality and quantity, temperature etc. [12]). At 20 °C the 138
development to adult (C6) takes approximately 10 days with the first brood appearing at 139
approximately day 14 [13]. This short development time from nauplii to adult indicate its suitability 140
for use in a short term sensitive developmental test for purported endocrine disrupting chemicals 141
that may affect normal copepod development (e.g. chitin synthesis inhibitors such as the 142
flubenzurons or moulting disruptors such as fenoxycarb). However, there are currently no existing 143
standardized regulatory test guidelines for developmental or reproductive studies with this 144
organism.
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Harpacticoids are widespread and are an important food source for macroinvertebrates and 146
fish. T. battagliai is a benthic copepod and harpacticoid copepods are often one of the most 147
dominant taxa in marine sediments [14], increasing their relevance as a test species for the 148
investigation of the effects of TEF and DIF. In addition, the sea lice, like T. battagliai, are also 149
members of the class Maxillopoda and subclass copepod, therefore they show similarities in their 150
development and lifecycle. Based on the MOA of the TEF and DIF, they are likely to affect the non- 151
target organism T. battagliai in a similar fashion to the main target organisms the sea lice. Figure 1 152
and Figure 2 describe the lifecycles of Lepeophtheirus salmonis (a problematic target species in 153
Norwegian aquaculture) and the test species T. battagliai, respectively.
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Due to the way in which these two compounds (DIF and TEF) are administered together in 155
fish farms in Norway, it is also of great importance that there is an understanding of potential 156
mixture effects in the marine environment and at the sediment water interface. For comparison 157
purposes, AZA was also assessed for acute toxicity and developmental effects with T. battagliai.
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However, due to its mode of action, it was predicted that at sub-lethal concentrations it would be 159
expected to have little effect on moulting and development. The selection of a developmental test 160
for use with T. battagliai was based on the need to assess possible chronic or developmental effects 161
of the chitin inhibiting flubenzurons (and the organophosphate pesticide azamethiphos) and to test 162
the hypothesis that non-target copepods could be affected in the same manner as the target sea lice 163
at environmentally relevant concentrations. The specific aims of the present study were (1) to look at 164
individual and mixture toxicity of TEF and DIF and AZA, (2) to develop an early stage naupliar 165
developmental test to investigate the sublethal toxic effects of flubenzurons and AZA on naupliar 166
growth and ecdysis and (3) make recommendations on improvements in study design and ease of 167
use, as well as contribute to the regulatory decision framework regarding the continued use of these 168
pesticides.
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MATERIALS AND METHODS 170
Test chemicals 171
The benzoylurea medicines, teflubenzuron (TEF) (CAS Registry No. 83121-18-0) and 172
diflubenzuron (DIF) (CAS Registry No. 35367-38-5) were obtained from Sigma-Aldrich Norway As. The 173
organophosphate medicine, azamethiphos (AZA) (CAS Registry No. 35575-96-3) was also obtained 174
from Sigma-Aldrich Norway As. The organic solvent dimethyl sulphoxide (DMSO) was used as a 175
solubilizing agent in the preparation of stock solutions of the benzoylurea compounds. Due to the 176
higher solubility of azamethiphos no solvent was required in the preparation of the stock solutions.
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Chemical analysis was conducted on the DMSO stocks for both TEF and DIF using LC/MS/MS analysis 178
to confirm the starting concentration of each test chemical [4].
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10 Acute toxicity of veterinary medicines to T. battagliai 181
All testing with T. battagliai was conducted with in house cultures. The acute toxicity testing 182
of all three test chemicals were conducted with slight modifications according to the ISO method 183
[15]. Toxicity tests were conducted with copepodids 6 ± 2 days old and with nauplii ≤ 3 h old. The 184
copepodid and naupliar tests were conducted in 12 well polystyrene plates (NUNC) and 48 well 185
polystyrene plates (NUNC) respectively. Four replicates, each containing 5 animals, were used with a 186
total of 20 animals per exposure concentration. All tests were incubated in a temperature controlled 187
room at 20 ± 2 °C and with a 16:8 h light:dark photoperiod. The lethality of each test chemical at 188
each concentration was recorded and the percentage mortality (LC50, lethal concentration of 50% of 189
the sample population) compared to the control was calculated after 24 and 48 h. Mortality is 190
defined as no swimming or appendage movements within an observation period of 10 seconds. At 191
test initiation and termination dissolved oxygen (DO), salinity and pH were measured to ensure 192
validity of the test. Measurements of these physico-chemical parameters were measured in the 193
controls, lowest and highest test concentrations. Seawater used in all testing was approximately 34 194
‰ and was obtained from the outer Oslofjord at a depth of ca.60 m.
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Optimisation of naupliar development test with T. battagliai 197
After initial acute toxicity determination with both copepodid and naupliar stages of 198
T battagliai, the optimization of a naupliar development test was conducted.
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The optimization trials included the use of various plates (12, 24 and 96 well polystyrene cell 200
culture plates), different feeding levels of the marine alga Rhodomonas baltica, and the use of 201
different renewal periods during a selected 7 day exposure period. For the final study design a 7 day 202
exposure period was selected to reflect a similar, environmentally relevant exposure scenario for the 203
organisms, based on the method of dosing of flubenzurons in Norwegian fish farms [4]. All trials were 204
conducted with natural filtered seawater in the absence of test chemicals at a salinity of ca. 34 ‰.
205
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Under optimal conditions, T. battagliai should pass through 5 naupliar stages and become copepodid 206
within 4 days, therefore, this time was used as the criteria for optimization of performance of the T.
207
battagliai in the various test designs. T. battagliai used in the trials were of a similar age as those to 208
be used in the definitive studies, ≤ 3 h old.
209
The Optimal food levels were assessed based on the survival and time to copepodid of 210
unexposed nauplii. The suitability of test plates and exposure volumes were assessed using survival 211
and general health as endpoints as well as developmental time.
212
213
Developmental effects of veterinary medicines to T. battagliai 214
Based on the optimization experiments a suitable test design for a 7 day naupliar 215
development study was selected, as follows. The selected test plates were 24 well polystyrene cell 216
culture plates (NUNC) with 2 mL of medium per cell well. T. battagliai ≤ 3 h old were individually 217
housed in 10 replicates per test concentration. Animals of the correct age were isolate by removing 218
ten gravid females from in house cultures several days before test initiation. These gravid females 219
were housed individually in approximately 5 mL of clean seawater containing food. Observations on 220
released offspring were made at hourly intervals until sufficient nauplii ≤ 3 h old could be collected 221
for use in the tests.
222
The animals were fed only once during the experiment and there was no requirement to 223
renew the medium during the exposure period. The food was prepared from a confluent healthy 224
culture of R. baltica that was settled for several days prior to test initiation. Once the R. baltica had 225
settled, the supernatant was poured off and the remaining algae resuspended in a minimum amount 226
of filtered seawater, mixed and a cell count was made using a Neubauer Improved (Bright-light) 227
chamber. Based on the total cell count a concentration of 2 x 105 cells/mL was fed to all test 228
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concentrations, controls and solvent controls. In order to reduce any dilution of the test compounds 229
once dispensed into the exposure wells, the R. baltica feed was prepared and spiked directly into 230
each of the test concentrations during preparation, prior to dispensing 2 mL per exposure well.
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Observations of mortality and developmental stage were made daily during the 7 day 232
exposure period. Time to copepodid and development rate were calculated for each individually 233
housed organism and the total number and percentage of copepods at the end of the study were 234
calculated per concentration and compared to the controls.
235
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Biological data analysis 237
All data (individual development rate, total number of copepods after 7 days, mortality and 238
EC values for total number of copepodids at day 7) were statistically evaluated with the commercial 239
software programme GraphPad Prism 6 for Windows and the EC values were calculated using the 240
Excel Macro REGtox. All calculations were performed using nominal concentrations.
241
Data for total number of copepodids after 7 days (numbers summed over all replicates within 242
a treatment) were analysed using Fischer’s Exact Test for 2 x 2 contingency tables. An overall 243
significance level of p = 0.05 was used. Mortality data (based on survivors) was assessed in the same 244
way for Day 7.
245
Individual development rates were calculated for each surviving organism. The development 246
rate is the reciprocal of the time to copepodid (reciprocal of the day number (day on which the 247
organism was copepodid) minus 0.5) and represents that proportion of naupliar development, which 248
takes place per day in the exposure system.
249
Statistical procedures were then used to look for significant differences in individual 250
development rates. In summary, data were tested for normality and homogeneity of variance prior 251
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to the use of a parametric (ANOVA followed by Dunnet’s post hoc test) or non-parametric (Kruskal 252
Wallis followed by Dunn’s post hoc test) procedures to identify significant differences between 253
treatments.
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RESULTS 256
Acute toxicity of the veterinary medicines to T. battagliai 257
The measured concentrations of the stocks used in all experiments were 1.5 mg/mL and 1.8 258
mg/mL for TEF and DIF respectively. At test termination for all acute studies, the DO concentrations 259
were greater than 4 mg/L at all measured concentrations. Salinity and pH were within ± 2 ‰ or ± 1 260
unit, respectively, throughout the tests. Control mortality for all acute tests was less than 10 % and 261
therefore all validity requirements for the standard ISO method were met.
262
The acute 48 h study with AZA and copepodids (as per ISO 14669) resulted in an LC10 and 263
LC50 (and corresponding 95 % confidence intervals) of 3.6 (2.6 – 4.5) µg/L and 7.7 (6.8 – 8.5) µg/L.
264
The same test with nauplii ≤ 3 h old at the start of the test, gave similar results of 3.4 (2.4 – 5.3) µg/L 265
and 6.7 (5.9 – 7.3) µg/L respectively for the LC10 and LC50values. Therefore based on these results a 266
range for the developmental study from 0.225 to 3.6 µg/L was selected.
267
For the flubenzuron compounds the acute data showed no effects up to 1000 µg/L for both 268
nauplii and copepodids with DIF. For the TEF acute studies there was no effect up to 1000 µg/L for 269
the copepodid however, the naupliar acute study with TEF proved more sensitive resulting in 24 and 270
48 h LC50 values of 230 (58 – 931) µg/L and 40 (4.8 - 419) µg/L. For acute studies with TEF and DIF a 271
limit of 1000 µg/L was used as the highest concentration. Even at these levels there is little 272
environmental relevance or realism about the concentrations used but it was deemed unnecessary 273
to test any higher as effects on other organisms have been observed at lower levels than those 274
tested in our experiments [16, 17]. In addition, these compounds are highly insoluble and are unlikely 275
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to occur in the environment in excess of these levels and are more likely to be found at ng/L levels in 276
marine waters [18].
277
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Optimisation of naupliar development test with T. battagliai 279
For the optimization of the test design for the naupliar developmental study, several 280
approaches were used. No test chemical was used in the study design experiments. Instead filtered 281
seawater, as would be employed as a negative control, was used for all experimental trials. The initial 282
proposed trial was based on the acute study test design, conducted in 12 well tissue culture plates 283
with a volume of ca. 5 mL per replicate. Ten individually housed animals per test concentration were 284
used instead of 4 replicates containing 5 animals, this was proposed so that individual development 285
could be tracked. After trialling a lower and higher feeding level, the final selected level for all trials 286
was based on previously published data (2 x 10 5 algal cells/mL Rhodomonas baltica) an optimal food 287
level for development [13, 19] of T. battagliai. Trials were performed for an initial 96 h, after which 288
time at optimal conditions all nauplii should become copepodid.
289
Due to the small size of the animals and the large volume of solution in the wells of the 12 290
well plates, it was difficult to find and assess the animals during the 96 h period. Originally the test 291
design also planned to incorporate an assessment of moulting on a daily basis. The assessment of 292
moulting proved difficult and not all moults could be accounted for within the wells. In addition, 293
within 96 h, under optimal conditions, they would have passed through all naupliar stages potentially 294
moulting more than once a day, therefore this observation was removed from the study design as 295
having limited value. In the 12 well test design with individually housed nauplii the survival was 100 296
% and all test organisms had developed to copepodid within the 4 days. The study was extended to 7 297
days to assess the need for renewal, during an extended exposure duration in the presence of a test 298
compound. The animals survived the 7 days and it was concluded that in this case there was no need 299
to renew the test medium. However, due to the large well size and volume of test medium (ca. 5 300
15
mL), it was time consuming to assess the animals, which were often found around the rim of the 301
wells, where visualization with the aid of a microscope proved difficult. It was therefore concluded 302
that the use of 12 well plates was not optimal for the assessment of naupliar development.
303
In the second experimental design, an assessment with 96 well plates was trialled. Due to the 304
size of the test organisms at the start of the test (≤ 3 h) and the small well size, it was possible to 305
assess the animals easily on a daily basis. The 96 well test design used ten individually housed 306
animals per test concentration. Each replicate well contained 200 µL of test medium containing algal 307
feed. This design was to incorporate a renewal period (on day 4) based on the small sample volume 308
and the hypothesis that there would not be enough surface area for sufficient gas exchange. Daily 309
observations were made and mortality, behaviour and any moults were observed. By day 3 the 310
animals were recorded as being in a poor condition. As T. battagliai are epibenthic they are often 311
observed on the bottom of the test wells. In the case of the 96 well test design the algae introduced 312
to the wells had quickly settled out on the small surface area on the bottom of the wells, where the 313
nauplii had become entangled. After 96 h there were no copepodids present, and after a further 24 h 314
all animals were dead. During the daily observations it did not appear that the animals were grazing, 315
instead they remained stationary, with movement only after gentle agitation. Another issue with the 316
96 well test design was generating sufficient volume at test termination for any required physico- 317
chemical analysis.
318
The final trial involved 24 well tissue plates containing 2 mL of test solution per replicate. All 319
other parameters were consistent with the previous two trials, feeding, medium (filtered seawater), 320
replication etc. No renewal was used in this trial. All animals were copepodid after 96 h and there 321
was only 10 % mortality (1 death) after 7 days. Therefore the use of the 24 well plates and all other 322
described parameters were used for the investigation of developmental effect of veterinary 323
pesticides to T. battagliai.
324
16 325
Developmental effects of veterinary medicines to T. battagliai 326
For the 7 day developmental effects investigations there were four testing scenarios using 327
the previously described 24 well test design: teflubenzuron only, diflubenzuron only, a 2:1 mixture of 328
teflubenzuron and diflubenzuron and azamethiphos only.
329
For all studies the DO, pH and salinity remained within acceptable limits and the survival in all 330
control and solvent controls was acceptable (≤ 10%).
331 332
Mortality data 333
The mortality data (day 7) were assessed and the acute NOEC and LOEC for 7 day mortality 334
for TEF were 0.0032 and 0.01 µg/L respectively. For DIF and the 2:1 TEF:DIF mixture the NOEC and 335
LOEC were 0.01 and 0.032 µg/L respectively. After 7 days exposure to AZA there was only 10 % 336
mortality at 3.6 µg/L, this was not statistically significant, therefore the LOEC was > 3.6 µg/L.
337 338
Total number of copepodids 339
The total number of copepodids on day 7 were compared to the control numbers for the four 340
different testing scenarios as per the mortality data. For TEF, significant differences in the number of 341
copepodids by day 7 was found between the control and 0.01 µg/L. It was not necessary to 342
statistically assess higher concentrations as there were significant mortalities at all concentrations 343
including 0.01 µg/L and above. Therefore, based on total number of copepods by day 7 the NOEC and 344
LOEC were 0.0032 and 0.01 µg/L respectively. For DIF, the NOEC and LOEC were 0.01 and 0.032 µg/L 345
respectively. For the 2:1 mixture, the NOEC and LOEC for total number of copepodids were the same 346
as for DIF, i.e. 0.01 and 0.032 µg/L. There were no statistically significant differences found for total 347
number of copepodids up to and including 3.6 µg/L for AZA.
348 349
Developmental rates 350
17
For individual development rates of each surviving animal (based on the day they were 351
recorded as copepodid) the data was assessed as previously describe. There were no statistically 352
significant differences in the individual development at any of the tested concentrations of AZA. For 353
TEF, the development rate of the individuals that successfully became copepodid was not 354
significantly different to the controls up to 0.0032 µg/L and at concentrations above this there were 355
either significant effects on the number of copepodids or on the number of mortalities (as described 356
previously). For DIF, the same was observed. At concentrations up to and including 0.032 µg/L, there 357
were no significant differences in individual development rates, however there were significant 358
differences in mortalities and number of copepodids by day 7 as previously described. The same 359
effect on individual development rate was observed with the 2:1 mixture, where all animals reached 360
copepodid at the same rate as the controls, however other endpoints such as mortality and number 361
of animals to reach the copepodid stage showed significant differences at low concentrations (Figure 362
3).
363 364
Summary of NOECs/LOECs 365
Time to copepodid, development rate, mortality and the total number of copepodids after 7 366
days exposure for all test scenarios are shown in Table 2. The overall NOEC/LOEC for the 367
developmental studies based on the most sensitive endpoints are 0.0032/0.01 µg/L (TEF, endpoints:
368
mortality and total number of copepodids at day 7), 0.01/0.032 µg/L (DIF, endpoints: mortality and 369
total number of copepodids at day 7), 0.01/0.032 µg/L (TEF:DIF, endpoints: mortality and total 370
number of copepodids at day 7). There was no effect compared to the control with any 371
concentration up to and including 3.6 µg/L for AZA with any endpoint.
372
DISCUSSION 373
The chitin synthesis inhibiting benzoylurea pesticides, teflubenzuron and diflubenzuron and 374
the organophosphate pesticide azamethiphos were all assayed with acute and developmental 375
18
studies with the non-target marine copepod, T. battagliai. The benzoylureas and azamethiphos were 376
selected due to their usage patterns in Norwegian aquaculture as well as the fact that they have 377
different methods for treatment (to the target organism) and different modes of action. TEF and DIF 378
are administered in fish feed, while AZA is applied as a bath treatment. TEF and DIF act by inhibiting 379
chitin synthesis in arthropods and AZA acts by the inhibition of cholinesterase activity. Based on 380
these points it was possible to hypothesis that TEF and DIF would be the most toxic in the 381
developmental assay and that although AZA may be acutely toxic [20, 21], it would be unlikely to 382
cause the same developmental effects as with the benzoylurea pesticides. This hypothesis is based 383
on the specific MOA of the benzoylurea pesticides that are specifically targeting organisms that 384
require chitin to develop through several stages to adult hood. Both the target organism (the sea 385
lice) and the non-target organism (T. battagliai) have similar lifecycles (Figure 1 and 2) and require 386
chitin in order to develop through several morphologically different life stages. Chitin inhibiting 387
chemicals may affect the moulting activity of these species by halting the process completely, by 388
retarding the process or by leaving the animals vulnerable to predation between moults. AZA on the 389
other hand is not likely to have an increased effect on T. battagliai development and was included to 390
act as a negative control (although acutely toxic) for developmental effects at environmentally 391
relevant levels in order to highlight the risk to specific groups of organisms through the increased use 392
of the flubenzuron pesticides.
393
As indicated previously, both TEF and DIF are applied in fish farms via the feed. The main 394
challenge with the treatment methodology is that a large amount of food will remain uneaten, Chen 395
et al. [22] estimated between 5-15 % of the administered food will be uneaten, and approx. 90% of 396
administered DIF and TEF will be excreted in the faeces [4]. DIF and TEF have low water solubility (89 397
µg/L and 9.4 µg/L respectively) and are relatively hydrophobic (Log Kow of 3.8 and 5.4 respectively) 398
and may therefore bind to particles and end up in the sediment. Due to its epibenthic nature, T.
399
battagliai is a relevant test species to assess the environmental hazard of these test substances 400
within the sediment as it is present at the sediment water interface and may be exposed to both 401
19
water soluble and particle bound contaminants. Due to limited amounts of data on DIF, the UK 402
environment agency does not have sediment Environmental Quality Standards (EQS) for this 403
substance. The Norwegian Environment Agency, is at present evaluating proposed sediment, water 404
and biota EQS values for both TEF and DIF [23]. The proposed EQSsediment = 0.2 µg/kg for DIF and 405
0.0004 µg/kg for TEF. Although our study focused on the assessment of toxicological effects to non- 406
target species through water only exposure systems, it is important to consider the risk to sediment 407
dwelling arthropods that may be affected in the same way as T. battagliai. The report of Langford et 408
al. [18] measured sediment concentrations of TEF and DIF as high as 269.2 and 136.6 ng/kg 409
respectively in the sediments around treated fish farms. The measured levels of TEF are in excess of 410
the new proposed sediment EQS values for Norway (EQSsediment =0.4 ng/kg) but not for DIF (EQSsediment
411
= 200 ng/kg). Therefore, there is a significant risk to organisms present in or on the sediment that 412
may be affected through the flubenzurons specific MOA of chitin synthesis inhibition.
413
In general, there is limited published ecotoxicity data on developmental and chronic effects 414
of benzoylurea pesticides on non-target species, especially data on marine organisms [18]. However, 415
in recent years several organizations have attempted to address the lack of data [7]. This is 416
particularly relevant in light of the recent resistance of sea lice to the commonly used emamectin 417
benzoate and the increased use of other aquaculture medicines such as the flubenzurons. Some 418
acute data for AZA exists and studies with lobster and mysid shrimp have yielded LC50 values of 1.39 419
µg/L (48 h) [24] and 0.52 µg/L (96 h) [25] while other species such as scallops and clams were 420
unaffected by AZA [26]. Therefore, from the results of the present study T. battagliai seems to show 421
a higher tolerance to AZA compared to other crustaceans.
422
Coppen and Jepson [27] described TEF as being more potent and toxicologically active than 423
DIF. This is apparent from the results of the present study where we have directly compared the two 424
pesticides, in both an acute and chronic test system, in which TEF consistently results as the more 425
toxic of the two. Surprisingly the toxicity observed with the 2:1 TEF:DIF mixture (reflecting treatment 426
20
with both medicines in Norwegian fish farms) elicited the same toxicological effects as DIF and not 427
TEF despite the TEF being present at a higher concentration in the mixture. As both TEF and DIF have 428
the same MOA, it has been suggested that the most likely description of the additive hazards from 429
these 2 chemicals will be concentration addition. In addition it has been suggested that testing of 430
mixtures of veterinary medicines should be conducted as though the organisms were exposed to 431
each compound independently. This is due to the fact that the active ingredients would normally not 432
be placed in the same product, if they compete for the same receptor target (i.e. have the same 433
MOA). Therefore, the chances of increased sensitivity to aquatic organisms due to a mixture effect, is 434
unlikely in the case of TEF and DIF [28]. As the mixture of the two chemicals was not more sensitive 435
than the results of the TEF alone, an assessment factor (AF) for the risk assessment purposes could 436
be applied to the NOEC from the TEF only developmental study. This would therefore be protective 437
of any potential mixture effects in the environment.
438
Recently published monitoring data for TEF and DIF from in and around fish farms in Norway 439
[18], treated with both TEF and DIF in combination and DIF alone, have shown elevated levels 440
present in seawater above the UK Environmental Quality Standards (EQS). Specific UK marine water 441
EQS values used for the assessment of the monitoring data where 5 ng/L (AA-EQS (Annual Average- 442
EQS)) and 100 ng/L (MAC-EQS (Maximum allowable concentration-EQS)) for DIF and 6 ng/L (AA-EQS) 443
and 30 ng/L (MAC-EQS) for TEF. The levels measured during the monitoring programme were in the 444
range of 34.3 – 295.2 ng/L (DIF) and < 1 – 12.9 ng/L (TEF), at the site treated with both DIF and TEF, 445
and 13.1 – 30.9 ng/L (DIF) for the site treated with DIF alone. These measured environmental levels 446
are higher than the developmental effect levels observed in the present study. The lowest effect 447
concentrations (LOEC) for T. battagliai development (number of copepodids on day 7) and mortality 448
were 10 ng/L and 32 ng/L for TEF and DIF respectively. Therefore it can be concluded that there is a 449
risk to non-target species in and around the areas treated with these pesticides. The recently 450
suggested environmental quality standards for sediments, water and biota (submitted to the 451
Norwegian Environment Agency) [23] propose EQSseawater values for TEF and DIF as follows. For DIF an 452
21
AA-EQS and MAC-EQS of 4 ng/L and 100 ng/L respectively have been suggested and for TEF an AA- 453
EQS and MAC-EQS of 2.5 ng/L and 12 ng/L have been put forward. These EQS values have only been 454
proposed to the Norwegian Environment Agency and have not officially been adopted by Norway.
455
With the specific MOA of flubenzurons it is important to consider the acute to chronic ratio 456
of these substances when performing environmental risk assessments. Flubenzurons elicit their 457
effect via chitin synthetase. Although chitin synthesis is not functionally part of the endocrine 458
system, enzyme systems that regulate chitin synthesis are sensitive to chemical alternation by 459
pesticides like teflubenzuron and diflubenzuron. Therefore they can be considered to be similar to 460
endocrine disruptors which specifically affect arthropods (and possibly other chitin producing 461
organisms). Endocrine disruptors typically have a very high acute to chronic ratio indicating that 462
although acute effects may not be seen at relatively high levels (e.g. mg/L concentrations) sub lethal 463
(chronic endpoints) can be observed at concentrations significantly lower (e.g. ng/L concentrations).
464
Historically, a large number of environmental risk assessments have involved estimating chronic 465
toxicity data from acute toxicity data using an assessment factor. Typically, the assessment factor has 466
been 100 resulting in a predicted chronic toxicity of 100 times lower than the LC/EC50. From a more 467
conservative point of view, some risk assessors have applied an assessment factor of 1000 which may 468
be considered more protective for the environment for substances with non-endocrine mediated 469
toxicity. For example, in terms of AZA which has been assessed in the present paper, the acute 470
toxicity was calculated at a concentration of between 6.7 and 7.7 µg/L dependent on the age of the 471
organisms. In addition, the chronic toxicity NOEC was calculated as 3.6 µg/L. This would mean that 472
the acute to chronic ratio is only a factor of 2 and by using an assessment factor based on the acute 473
toxicity data alone would have provided adequate protection for T. battagliai. However, considering 474
the flubenzurons assessed within the present study and specifically the most toxic (TEF) the acute to 475
chronic ratio is significantly higher than the assessment factor approach would have predicted the 476
chronic toxicity. For example, the acute toxicity value for TEF was >1000 µg/L and the NOEC was 477
0.0032 µg/L which results in an acute to chronic ratio >312500.
478
22
As a preliminary risk assessment for both TEF and DIF, a PNEC has been calculated based on 479
the NOEC values of 0.0032 and 0.01 µg/L respectively from the T. battagliai naupliar developmental 480
tests. An AF of 10 was applied to both these values to derive the PNEC. Justification for the selection 481
of this AF is based on an assessment of the sensitivity of the endpoint, existing EQS values and 482
detection limits for DIF and TEF (1 ng/L, [4]) in water. Therefore, the PNECseawater for TEF and DIF 483
would be 0.32 and 1.0 ng/L respectively. Comparing these PNEC values with the Measured 484
Environmental Concentration (MEC) from Langford et al., [18] to derive Risk Quotients (RQ) for TEF 485
and DIF would indicate that an even higher number of the monitored sites would be in exceedance of 486
the RQ of 1 than the previous conclusion [18] based on using the existing EQS values.
487
488
RECOMMENDATIONS AND CONCLUSIONS 489
In recent years, several naupliar developmental studies with marine copepods have been 490
developed and now exist as ISO draft standards (ISO/DIS 16778 [29], ISO/TC 147/SC 5 N 761 [30].
491
These draft standards assess the developmental effects of Acartia tonsa and Nitocra spinipes from 492
nauplii to copepodid. These studies are approximately the same length as the proposed T. battagliai 493
method. However, with the latter, the controls should be copepodid by 96 h, therefore the length of 494
time for the test could be reduced to just 4 days, compared to 6-7 days or 5-6 days for the N. spinipes 495
and A. tonsa studies. The idea behind extending the T. battagliai test described in the present study, 496
is to allow for the observation of retardation of development, where the end point may not be that 497
the animals do not develop, rather that it happens at a slower developmental rate compared to the 498
control. However, if the aim is to look only at number of copepodids at the point where > 80 % of the 499
controls have developed to C1, then 96 h is an acceptable time period for the test. In addition, 500
another benefit of the proposed T. battagliai test is that individual development rate can be tracked, 501
which is not the main consideration in the aforementioned ISO standards. Possible improvements to 502
the proposed study design are an increased number of replicates or increased number of test 503
23
concentrations (> 5) although the ease of testing with this study design and the minimal number of 504
animals required, that still provides a reasonably robust statistical assessment, makes this method 505
ideal for assessing chronic toxicity in copepods.
506
In conclusion, the results of the investigations into the acute and toxic effects of the 507
flubenzurons (DIF and TEF) and AZA have indicated distinctly different effect patterns between the 508
two types of veterinary medicines. Azamethiphos was acutely toxic to the test organism T. battagliai 509
as has been previously described in the literature to other crustaceans. However, at lower levels, 510
there were no observed developmental effects. In contrast, the flubenzurons, displayed little or no 511
acute toxicity at microgram per litre levels over a period of 48 h and developmental effects were 512
seen in the nanogram per litre range. The latter reflects the potential for adverse effects at 513
environmentally relevant concentrations to non-target organisms within the marine environment.
514
Taking into consideration the extremely high acute to chronic ratio of flubenzurons, underpins the 515
importance of designing tests appropriately to the specific MOA. If the environmental risk 516
assessment had been performed on acute toxicity data alone, there would have been a significant 517
discrepancy in the risk quotient. This may have resulted in flubenzurons not being regulated 518
appropriately or sufficiently enough within the aquaculture industry and other substances with 519
similar modes of action should be considered correspondingly for environmental risk assessment 520
purposes.
521
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31. Mattilsynet, 2014. Lakselusmiddelforbruket økte også I 2013. Publisert 4.3.2013. [cited 2014 637
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13.12980 640
641
30 642
List of Figure legends:
643 644
Figure 1. Lifecycle of Lepeophtheirus salmonis modified from Schram (1993) with adaptations based 645
on Herme (2013) 646
647
Figure 2 Lifecycle of Tisbe battagliai modified from Hutchinson et al (1999) with adaptations from 648
Volkmann-Rocco (1972) 649
650
Figure 3. Development rate (± standard deviation) of nauplii (N1) to copepodid (C1) for (a) 651
Diflubenzuron, (b) Teflubenzuron, (c) TEF:DIF, (d) Azamethiphos. * indicates statistical difference 652
compared to the control.
653
654 655
Fig 1
Fig 2
Fig 3
Table 1. Selected products used against sea lice in aquaculture in Norway. (kg of active substance).
(Source: Mattilsynet 2014)
Active substance
2004 2005 2006 2007 2008 2009 2010 2011 2012 2013
Azamethiphos 66 1884 3346 2437 4059 3037
Diflubenzuron 1413 1839 704 1611 3264
Teflubanzuron 2028 1080 26 751 1704
Emamectin 32 37 60 73 81 41 22 105 36 51
Table 2 Summary of Tisbe battagliai development and mortality data for all test compounds
Concentration (µg/l)
Time to first copepod (days)
Mean time to first
copepodid (days)
Number copepodid (day 7)a
Mortalities Day 7 (%)a
Mean development Rate (days-1)b
TEFLUBENZURON
Control 3 3.8 10 0 0.314
Solvent control 3 3.6 10 0 0.337
0.0032 3 3.9 9 10 0.335
0.01 5 5 1 80 NA
0.032 - - 0 90 NA
0.1 - - 0 100 NA
0.32 - - 0 100 NA
DIFLUBENZURON
Control 4 4.8 9 10 0.239
Solvent control 5 5.0 9 10 0.222
0.0032 5 5.1 9 10 0.218
0.01 5 5.3 8 20 0.189
0.032 5 5.5 3 70 0.202
0.1 - - 0 90 NA
0.32 - - 0 100 NA
2:1 Mix TEF:DIF
Control 4 4.4 10 0 0.260
Solvent control 3 4.2 10 0 0.277
0.0032 3 4.6 9 10 0.264
0.01 4 5.1 9 10 0.225
0.032 5 5.8 4 50 0.195
0.1 - - 0 50 NA
0.32 - - 0 90 NA
AZAMETHIPHOS
Control 3 3.1 10 0 0.389
0.225 3 3.4 10 0 0.359
0.45 3 3.1 10 0 0.389
0.9 3 3.1 10 0 0.389
1.8 3 3.2 10 0 0.377
3.6 3 3.1 9 10 0.387
b Nominally 10 animals tested per concentration.
a individual development rate = 1/(day number-0.5). All developmental rates are given to three significant figures.
NA = Not applicable
